ABSTRACT Solid tumors contain diverse cellular ecosystems, and this heterogeneity has been implicated as a key factor driving disease progression, metastasis, and drug resistance. Hence, identifying the presence and function of each cell subtype is needed to fully understand tumor drivers and guide therapeutic decisions. Increasingly, single cell analysis methods are being used to define cellular subsets within tumors to address biological and therapeutic questions. A method that is rapidly emerging is single cell RNA sequencing (scRNA-seq), which enables identification of cell type and function based on the transcriptome. However, tumor tissue must first be converted into single cells, and this step is a critical barrier to more widespread use of single cell analysis methods, particularly in clinical settings. Standard tissue dissociation protocols involve several manual steps that are slow, inefficient, and highly variable. Commercial systems only accomplish part of the workflow and are not well-characterized. In previous work, we developed novel microfluidic digestion, dissociation, and filtration devices that utilized hydrodynamic forces to break down tissue into single cells. We recently combined the devices into a platform and attained excellent results for various murine tissues, including breast tumors. Specifically, the device platform significantly enhanced single cell recovery and decreased cell subtype biasing. To date, these devices have not been applied to human tumor specimen. In this proposal, we will develop a powerful tumor dissociation platform that can perform the entire workflow in a fast, efficient, and gentle manner. We will focus on creating a prototype benchtop fluidic system that contains all of the necessary components (i.e. pumps, valves, monitoring capabilities, control software) to operate the microfluidic cartridges in an optimal and automated manner. We will first create the system and perform basic feasibility and quality control tests. We will then optimize performance of the fluidic system and microfluidic cartridges using a murine breast tumor model, with single cells analyzed by flow cytometry and scRNA-seq. As part of this work, we will also increase tissue sample size so that entire tumor resections can be processed. Finally, we will perform a small validation study using human breast tumor specimens. The Specific Aims for this 6 month project include (1) create the prototype fluidic system, (2) optimize fluidic system using murine tumor tissue, and (3) validate performance using human breast tumor specimen. The single cell analysis market is expected to grow robustly, driven by growing biotechnology and pharmaceutical industries, rise in single cell genomics, and applicability to cancer for basic research and personalized medicine. Our microfluidic dissociation technologies are poised to make a significant impact on the single cell analysis of tumor tissues by making it easier and more reliable to prepare s...